Detection of potentially enterotoxigenic staphylococci is an important aspect of food processing, and may be used as a means of screening for indications of contamination during processing and for post-processing contamination. Food sample evaluations for potentially enterotoxigenic staphylococci can serve as a direct indication of the presence of potential pathogenic species in food. The detection of Staphylococcus aureus (S. aureus), a known enterotoxigenic species, is especially important in food processing. Other potentially enterotoxigenic species of Staphylococcus are known and the testing of samples for contamination with these species also may be important. In addition, the testing of patient samples to indicate possible pathogenic staphylococcal infection is of importance in the clinical setting.
One method for testing a sample for the presence of staphylococci includes a thin film culture device that includes a dry, reconstitutable culture medium. The culture medium includes a two-indicator system that provides differential colony staining after about 24-40 hours of incubation. Staphylococci in the sample produce metabolites that react with one indicator, a phosphatase substrate, to produce red or red-violet colonies. Non-staphylococci bacteria produce metabolites that react with the second indicator, a glucopyranoside substrate, to produce blue colonies. This method cannot distinguish between S. aureus and other staphylococci.
One current method for detecting S. aureus uses Baird-Parker egg yolk-tellurite-pyruvate agar medium (abbreviated as BPA) for determining the presumptive presence of S. aureus in a fractional part of a sample. In this method, BPA plates are examined for the presence of “typical” and “a typical” colonies after 48 hours incubation. Samples of the colonies are then transferred to brain heart infusion for an additional incubation of up to 24 hours. The broth cultures are mixed with rabbit plasma for an additional 6-24 hours incubation. The culture-plasma mixtures are then evaluated for the presence of coagulation of the plasma (i.e., clotting). Cultures giving rise to a clot are considered to be coagulase positive. A presumptive positive from BPA followed by a coagulase-positive result is considered to be confirmation of the presence of S. aureus in the sample.
The use of coagulase activity associated with the presence of S. aureus also has been thought to correlate with potential pathogenicity, including enterotoxin production. The tedious, time-consuming nature of the coagulase test, however, makes it impractical for routine testing of large numbers of samples.
Two alternatives to the coagulase test have shown good statistical relation to the coagulase reaction of S. aureus: hyaluronidase and thermostable nuclease (TNase). The hyaluronidase system, however, is complex and costly. Testing for TNase activity was also tedious until Lachica et. al., Applied Microbiology 21(4), pp. 585-87 (1971), described the use of the metachromatic dye, toluidine blue O (TBO), for the detection of TNase by the differential staining in the presence of hydrolyzed and unhydrolyzed DNA.
The TNase detection method has been described and used in methods including (1) forming wells in a TBO/DNA agar-filled petri dish and placing boiled cultures within the well to determine the presence of TNase, (2) forming wells in a TBO/DNA agar medium cast on the surface of a microscope slide (or equivalent) and following the procedure of (1), (3) overlaying a Baird-Parker agar (or equivalent) plate with molten TBO/DNA agar after the developed BPA plate has been pre-incubated at 60° C. for at least 2 hours. (1), (2), and (3) give readable results in 2-4 hours from colonies or suspensions that are positive for TNase. Using these methods, various investigators have shown correlation of the TNase test with the coagulase test for S. aureus of up to 100%.
Enterotoxigenic staphylococci also may be detected using an article containing unhydrolyzed nucleotides and toluidine blue O that is adapted for placement against a sample suspected of containing enterotoxigenic staphylococci. The sample must be pre-heated to 60° C. for at least about 30 minutes to inactive non-thermostable nuclease activity. If TNase is present, which correlates to the presence of enterotoxigenic staphylococci, it will hydrolyze nucleotides provided in the article. The hydrolyzed nucleotides will react with the toluidine blue O to produce a detectable red or pink halo surrounding the colony containing the enterotoxigenic staphylococci.
TNase activity also has been detected in other potentially enterotoxigenic Staphylococcus species, including some that are coagulase negative.
While some methods of TNase testing are reliable, their utility in testing or screening large numbers of samples is severely limited by the need to form wells or prepare molten agar in order to obtain results, which are time consuming and inefficient techniques in the context of testing large numbers of samples. It would thus be desirable to develop a TNase test for potentially enterotoxigenic staphylococci that would permit efficient and reliable testing or screening of large numbers of samples, in food processing or in clinical applications.